The present invention relates to a vector having a nucleic acid insert coding for a mutated prion protein, a mutated prion protein, a DNA coding for such a protein and a process for the preparation thereof. Furthermore, this invention concerns antibodies, a vaccination agent and the use of the above-mentioned subject matters. Moreover, the present invention relates to non-human mammals, e.g. working animals, which are resistant to prion infections.
The prion diseases which have spread in the past few years and include inter alia BSE, Creutzfeldt-Jakob disease and scrapie, represent a group of absolutely fatal neurodegenerative diseases in human beings and animals which are caused by infectious pathogens whose structure is still unclear and which are referred to as xe2x80x9cprionsxe2x80x9d below. For the time being there are no satisfactory possibilities regarding the prophylaxis and/or treatment of these diseases. The development of such prophylaxis/treatment possibilities is made more difficult because the molecular pathogenesis of prion diseases is presently not clarified to a sufficient extent. Only a slight delay of the prion replication has been achieved by means of various drugs so far, but the infection could by no means be brought to a stand-still (Priola et al., Inhibition of scrapie-associated PrP accumulation. Probing the role of glycosaminoglycans in amyloidogenesis, Mol. Neurobiol. 8, pp. 113-120 (1994). Furthermore, such a treatment is accompanied by considerable side-effects, which is due to the fact that these drugs only have a very unspecific effect.
Therefore, it is the object of the present invention to provide a product serving for rendering working animals whose tissues or products are used as foodstuffs, drug constituents or cosmetics for human use resistant to prion propagation and protecting infected human or animal individuals from the onset of the infection and the progression thereof, respectively.
According to the invention this is achieved by the subject matters defined in the claims.
Thus, the subject matter of the present invention relates to a vector containing a nucleic acid insert coding for a mutated prion protein, the mutated prion protein having an insertion, substitution and/or deletion within the H1 region. It should preferably be a vector suitable for gene prophylaxis and gene therapy, respectively.
On the basis of this invention the inventors modified critical regions, e.g. the Helix1 region (H1) (Gasset et al., Predicted alpha-helical regions of the prion protein when synthesized as peptides form amyloid, Proc. Natl. Acad. Sci. USA 89, p. 10940, 1992) in the prion protein by means of genetic technique by carrying out e.g. mutations in the form of insertions, substitutions or deletions, particularly deletions. In this connection, the inventors found out that these modified recombinant proteins lose their capability of being convertible into the amyloid-like, disease-associated form of the prion protein (PrPSc). In addition, it was found that the presence of the mutated proteins has an inhibitory effect on the conversion of the endogenous wild-type prion protein (PrPc) which is naturally present in the organism into the disease-associated form (PrPSc) (what is called dominant-negative effect).
The expression xe2x80x9cvector suitable for gene therapyxe2x80x9d comprises any vector which is suitable for the gene transfer, i.e. the introduction of nucleic acids into cells. The vector may remain episomally within the cells or be integrated into the genome. Furthermore, the vector may be a plasmid vector or viral vector. Examples of viral vectors are retroviral, herpes simples virus, cytomegaly virus (CMV), adenovirus, vaccinia virus or adeno-associated virus (AAV) vectors. pUC18 or pUC19 have to be mentioned as preferred plasmid vectors.
The expression xe2x80x9cmutated prion proteinxe2x80x9d comprises a prion protein which as compared to the wild-type has been modified as regards its function and/or its length by means of suitable measures with which a person skilled in the art is sufficiently familiar. This may be done e.g. by inserting mutations on a nucleic acid level, e.g. by carrying out insertions, substitutions and particularly deletions. Methods of producing the above modifications within the nucleic acid sequence are known to the person skilled in the art and described in standard works of molecular biology, e.g. in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. (1989). The mutation is carried out within the H1 region of the prion protein within which an accumulation of identical amino acids occurs (Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala) and which is conserved with all animal species. As to the sequences of the prion proteins of various organisms reference shall be made to Prusiner, S. B., Molecular structure, biology and genetics of prions, Adv. Virus Res. 35, p. 83 et seq. (1988); Schxc3xa4tzl, H. M. et al., Prion protein gene variation among primates, J. Mol. Biol. 245, p. 362 et seq. (1995) and Gabriel, J. M. et al., Molecular cloning of a candidate chicken prion protein, Proc. Natl., Acad. Sci. USA 89, p. 9097 et seq. (1992). In a particularly preferred embodiment, the modification concerns amino acids 108-121 (based on the mouse PrP sequence), preferably 112-121, most preferably 114-121. This modification must usefully cover more than one amino acid, i.e. at least two, since otherwise the described properties such as the non-convertibility into the disease-associated form of the prion protein (PrPSc) and/or the trans-dominant inhibition of the accumulation of wild-type PrPSc cannot be expected. Since as proved the region of amino acids 112 to 121, preferably 114 to 121, contains the strongest amyloidogenic amino acids of the prion protein, a deletion is the preferred route of preparing a modified protein comprising the above-mentioned properties. Following the deletion of at least two of the amyloidogenic amino acids in the above-mentioned region, another route is the substitution by amino acids which are known to have no tendency of forming a xcex2-pleated sheet structure or can even destroy an existing xcex2-pleated sheet structure (e.g. proline). The direct insertion of amino acids which counteract the formation of a xcex2-pleated sheet structure, e.g. proline, is also conceivable. Another advantageous modification of the prion protein is made at the carboxy terminus. The already spontaneously occurring secretion of PrP molecules is enhanced by this modification (Borchelt et al., Release of the cellular prion protein from cultured cells after loss of its glycoinositol phospholipid anchor, Glycobiology 3, pp. 319-329 (1993). This is achieved according to the invention in that mutated prion proteins are secreted to an increased extent by the producing cells by a lack of glycoinositol phospholipid anchor. Thus, it is possible to reach by diffusion also those cells in spatial neighborhood which do not express the mutated prion protein per se. As a result, the desired dominant negative effect is expanded to a much greater number of target cells.
The expression xe2x80x9cnucleic acid insertxe2x80x9d comprises any nucleic acid such as DNA or RNA which codes for a mutated prion protein. It is favorable for the nucleic acid insert to be expressible, e.g. when it is controlled by a constitutive or inducible promoter. A nucleic acid insert in the form of a DNA which comprises.the following is preferred:
(a) the DNA of FIG. 4 or a DNA differing therefrom by one or several base pairs,
(b) a DNA hybridizing with the DNA from (a), or
(c) a DNA related to the DNA from (a) or (b) via the degenerated genetic code.
The expression xe2x80x9chybridizing DNAxe2x80x9d refers to a DNA which hybridizes with a DNA from (a) under conventional conditions, particularly at 20xc2x0 C. below the melting point of the DNA. In this connection, the term xe2x80x9chybridizingxe2x80x9d refers to conventional hybridization conditions, preferably to hybridization conditions where 5xc3x97SSPE, 1% SDS, 1xc3x97Denhardts solution are used as the solution and the hybridization temperatures are between 35xc2x0 C. and 70xc2x0 C., preferably 65xc2x0 C. After the hybridization washing is preferably carried out first with 2xc3x97SSC, 1% SDS and then with 0.2xc3x97SSC at temperatures between 35xc2x0 C. and 70xc2x0 C., preferably at 65xc2x0 C. (for the definition of SSPE, SSC and Denhardts solution see Sambrook et al., supra). Stringent hybridization conditions as described e.g. in Sambrook et al., supra, are particularly preferred.
According to the invention an above nucleic acid insert is inserted in an above vector. The insertion is made such that the nucleic acid fragment can be expressed. This can be achieved by inserting in phase the nucleic acid fragment in an expression unit present in the vector. To this end, it may be necessary to at least partially remove a DNA present in the expression unit. It may also be advantageous to replace elements of the existing expression unit, such as enhancers, promoters or polyadenylation site, at least partially by others. A promoter which is specific to a tissue kind is preferably inserted in an expression unit, so that the expression of the nucleic acid insert which is controlled by the promoter becomes tissue-specific. The above nucleic acid insert can also be expressed in an expression unit which must be introduced into the vector for this purpose. In order to express the mutated prion protein according to the invention, the vector is transformed into suitable cells. Examples of such cells comprise the E. coli strains HB101, DH1, x1776, JM101, JM109, BL21 and SG13009, the yeast strain Saccharomyces cerevisiae and the animal cells Neuro 2a, 3T3, FM3A, CHO and COS. Moreover, the person skilled in the art is familiar with conditions of culturing transformed cells and transfected cells, respectively. He also knows methods of isolating and purifying the protein expressed by the nucleic acid insert (particularly by the DNA). Such a protein and fragments thereof also form a subject matter of the present invention.
In the case of virus vectors, it often proves to be favorable to insert the nucleic acid fragment in an expression unit present in the vector. The possibly accompanying removal or partial removal of virus DNA present in the expression unit then results in a virus vector which has a defect in a viral function. This defect can be used as a selection marker. On the other hand, the defect can be compensated, if necessary, by common methods such as complementation in trans. Virus vectors in which the nucleic acid fragment is inserted such that the virus vectors per se are no longer capable of forming infectious wild-type viruses are preferred according to the invention.
Common methods can be carried out for the preparation of an above vector. In a preferred embodiment, the vector according to the invention is pUC-PrPxcex94H1 or pCMV-PrPxcex94H1. The latter was deposited as E. coli culture with the DSMZ Braunschweig (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH [German-type collection of microorganisms and cell cultures], Mascheroder Weg 1b, 38124 Braunschweig) under number DSM 11400 on Feb. 6, 1997. The preparation of the pCMV-PrPxcex94H1 vector is described in Example 1 below. The PrPxcex94H1 (SEQ ID NO:1) insert used for this purpose (nucleic acid fragment) originates from the mouse prion protein gene and codes for a mutated protein which is 8 amino acids shorter than the wild type. The deletion was inserted in the wild-type gene by means of site directed mutagenesis and is found from positions 438 to 461 (based on the counting according to Locht et al., Proc. Natl. Acad. Sci USA 83, p. 6372 (1986), i.e. within the H1 region of the prion protein and relates to amino/acids (Ala-Ala-Ala-Ala-Gly-Ala-Val-Val [amino acids 114-121](SEQ ID NO:8). A sequence comparison between the mouse wild-type gene and the employed insert PrPxcex94H1 is shown in FIG. 2 (SEQ ID NO: 2). The DNA sequence of PrPxcex94H1 is shown additionally in FIG. 4 (SEQ ID NO: 1). The second above-mentioned vector pUC-PrPxcex94H1 according to the invention was prepared by cloning the PrPxcex94H1 insert according to FIG. 4 (SEQ ID NO: 1) into pUC19 via AatII/SacI.
A further subject matter of the present invention relates to an antibody directed against an above protein. Such an antibody can be prepared by common methods. It may be polyclonal or monoclonal. For its preparation it is favorable to immunize animalsxe2x80x94particularly rabbits or chickens for a polyclonal antibody-and mice for a monoclonal antibodyxe2x80x94with an above protein or with fragments thereof. Further boosters of the animals can be made with the same protein or with fragments thereof. The polyclonal antibody can then be obtained from animal serum and egg yolk, respectively. For the preparation of the monoclonal antibody animal spleen cells are fused with myeloma cells.
The above subject matters are particularly suitable to provide a prophylaxis and/or treatment in the case of prion diseases. In this connection, one makes use of the effect that the mutated prion proteins cannot be converted into the infectious form, on the one hand, and that by overexpression of the mutated prion proteins existing cellular wild-type prion proteins (PrPc) are prevented from converting into PrPSc, on the other hand. In the latter case, it is recommended that the PrP mutation be carried out on the PrP DNA sequence of the target species analogously to ensure the strongest possible protein-protein interaction between PrPc and PrPSc which is necessary for the desired inhibitory effect. Prophylaxis of prion diseases is effected e.g. by the production of transgenic working animals which carry a mutated PrP gene either exclusively or in addition to the endogenous wild-type PrP gene. A person skilled in the art knows suitable transgenesis methods. For example, efficient methods of producing transgenic cattle by the insertion of foreign (additional) genes are available (Hyttinen et al., xe2x80x9cGeneration of transgenic dairy cattle from transgene-analyzed and sexed embryos produced in vitroxe2x80x9d, Biotechnology N.Y., 12, pp. 606-608, 1994). In addition, reference is made to the mechanism of the homologous recombination (cf. R. M. Torres, R. Kxc3xchn, Laboratory Protocols for Conditional Gene Targeting, Oxford University Press, 1997) in embryonic stem cells (e.g. mouse 129/SV) and new and obviously very efficient nuclear transfer technology (E. Pennisi, xe2x80x9cAfter Dolly, a Pharming Frenzyxe2x80x9d, Science 279, pp. 646-648, 1998), respectively. The homologous recombination between the DNA sequences present in a chromosome and the new, added cloned DNA sequences enables the insertion of a cloned gene in the genome of a living cell in place of the original gene. By means of this method it is possible to obtain animals which are homozygotic for the desired gene or the desired gene portion or the desired mutation, via chimeras using embryonic stem cells.
Furthermore, double-transgenic mammal models can be produced by crossbreed of the transgenic mammal according to the invention with already established transgenic mammals.
A gene substitution vector (e.g. on the basis of the above described plasmid pCMV-PrPxcex94H1) and an embryonic stem cell line of the corresponding non-human mammal (ES 129/SV is preferred in the case of a mouse, for example) are used for the production of a transgenic mammal according to the invention via the mechanism of the gene exchange. In this connection, the resulting recombinant clones of the embryonic stem cells are injected into blastocytes which are then implanted into suitably prepared female animals for the production of chimeras. Here, xe2x80x9csuitably prepared female animalsxe2x80x9d means that the animals have been prepared by various measures, e.g. by a hormone treatment, for their task of carrying to full term. In this connection, reference is also made to xe2x80x9cR. M. Torres, R. Kxc3xchn, see abovexe2x80x9d. Since it is advantageous to have available homozygotic animals, the resulting heterozytogic strains can then be mated with another animal of the same strain or another (heterozygotic) strain. Thus, a homozygotic condition can be reached by cross-breed. This serves for producing prion-resistant animals which can subsequently be further bred according to standard methods.
The gene therapy of prion diseases in human or animal individuals can be made by transfection and transduction, respectively, of somatic cells of the corresponding organism, such as neurons, gliacytes, fibroblasts or cells of the cerebral blood vessels, with the vector according to the invention. The transduction can be carried out according to common methods. If the vector is present as viral particle, it will be favorable to infect the cells therewith. On the other hand, if it is present as a nucleic acid, e.g. DNA, it will be advisable to transfect the cells therewith. For example, electroporation, calcium-phosphate co-precipitation, lipofection and particle gun have to be mentioned as transfection techniques. The cells may be present in the organism. On the other hand, the cells to be transduced can also be isolated from the organism, be transduced outside the organism and returned into the organism again. Such cells are referred to as autologous cells. In addition, it is also possible to use allogenic cells for the transduction as regards the organism. In this connection, it is favorable for these cells to belong to an HLA type corresponding to the organism.
A further subject matter of the invention relates to a vaccination agent which comprises an above vector, an above protein or fragments thereof and conventional auxiliary agents, such as buffers, diluents, carriers, etc.
By means of the present invention it is possible to stop the onset of a prion infection within the meaning of a prophylaxis and prevent the progression of such infections in already infected individuals, respectively. On the one hand, it is possible to eliminate the risk of an infection of human beings via the food chain, via animal-derived pharmaceutical preparations and cosmetics, respectively, by the production of transgenic working animals (e.g. cattle, sheep, goats, pigs, chickens, fish) which by carrying the mutated PrP gene are protected from a prion infection from the very beginning. On the other hand, the present invention enables to prevent and treat, respectively, an otherwise fatal disease via an in vivo gene therapy and ex vivo gene therapy, respectively.
As compared to a conventional treatment, a gene therapy/prophylaxis has various advantages which are based on the specificity of the molecular point of attack (e.g. interference in the protein-protein interaction between PrPc and PrPSc). The freedom from side-effects and simultaneous high degree of efficiency also result therefrom. A gene therapy/prophylaxis can be made as follows:
1) Production of prion-resistant working animals: the vector according to the invention serves for producing transgenic animals which in place of the endogenous wild-type PrPc or in addition to the endogenous wild-type PrPc, express a mutated prion protein which per se can no longer be converted into a protease-resistant PrPSc form and can additionally prevent the conversion from wild-type PrPc into PrPSc. As compared to transgenic xe2x80x9cknockoutxe2x80x9d animals, i.e. animals which express no prion protein at all, the transgenic animals which express the mutated form have the advantage that no phenotypic differences occur with respect to xe2x80x9cnormal animalsxe2x80x9d, since animals having a mutated prion protein modified in only one small region certainly still contain the normal physiological functions of the prion protein. The infection of these transgenic animals and thus another spread of e.g. the BSE epidemic would be prevented in working animals by means of this gene prophylaxis.
2) Artificial insemination/in vitro fertilization: sperm from a transgenic prion-resistant working animal, e.g. cattle, is used to produce further generations of resistant working animals by means of the artificial insemination route common for breeding working animals today. An in vitro fertilization is also considered preferably, since it offers the advantage of investigating the embryos for their genetic state, particularly the presence of the transgene, even before they are implanted into the uterus.
3) ex vivo gene therapy: Autologous cells from a patient, which can be implanted e.g. into the central nervous system are removed and transfected and transduced, respectively, with the vector according to the invention as cell culture and reimplanted into the patient.
4) in vivo gene therapy: A vector according to the invention (e.g. a recombinant virus) with an open reading frame of a mutated prion protein and suitable control elements (e.g. promoters, enhancers, polyadenylation signal, etc.) is used to directly infect the affected cells (e.g. nerve cells) thus preventing the spread of a disease within the CNS.